Alpha-2A adrenergic receptor (ADRA2A) iRNA agent compositions and methods of use thereof
The disclosure relates to double stranded ribonucleic acid (dsRNAi) agents and compositions targeting an alpha-2A adrenergic receptor (ADRA2A) gene, as well as methods of inhibiting expression of an ADRA2A gene and methods of treating subjects having an ADRA2A-associated disease or disorder, e.g., a primary tauopathy or Alzheimer's disease, using such dsRNAi agents and compositions.
1 . A double stranded ribonucleic acid (dsRNA) agent for inhibiting expression of ADRA2A, wherein the dsRNA agent comprises a sense strand and an antisense strand forming a double stranded region,
wherein the antisense strand comprises a region of complementarity to an mRNA encoding ADRA2A, and wherein the region of complementarity comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from SEQ ID NO: 153 or 423.
2 . The dsRNA agent of claim 1 , wherein the sense strand comprises at least 15 contiguous nucleotides differing by no more than three nucleotides from any one of the nucleotide sequence of nucleotides 528-548 of SEQ ID NO: 1, and the antisense strand comprises at least 15 contiguous nucleotides from the corresponding nucleotide sequence of SEQ ID NO: 2; and/or
the antisense strand comprises at least 15 contiguous nucleotides differing by no more than three nucleotides from the antisense strand nucleotide sequences of a duplex AD-1201752.
3 . The dsRNA agent of claim 1 , wherein the sense strand comprises SEQ ID NO: 18 and the antisense strand comprises SEQ ID NO: 153, or the sense strand comprises SEQ ID NO: 288 and the antisense strand comprises SEQ ID NO: 423.
4 . The dsRNA agent of claim 1 , wherein the sense strand, the antisense strand, or both the sense strand and the antisense strand is conjugated to one or more lipophilic moieties, optionally wherein:
the lipophilic moiety is conjugated to one or more internal positions in the double stranded region of the dsRNA agent;
the lipophilic moiety is conjugated via a linker or carrier; and/or
lipophilicity of the lipophilic moiety, measured by logKow, exceeds 0.
5 . The dsRNA agent of claim 4 , wherein the one or more lipophilic moieties are conjugated to one or more internal positions in the double stranded region of the dsRNA agent on at least one strand, optionally wherein:
(i) the one or more lipophilic moieties are conjugated to the one or more internal positions on at least one strand via a linker or carrier, optionally wherein:
(a) the internal positions include all positions except the terminal two or three positions from each end of the at least one strand; and/or
(b) the internal positions exclude a cleavage site region of the sense strand, optionally wherein the internal positions include (i) all positions except positions 9-12, counting from the 5′-end of the sense strand or (ii) all positions except positions 11-13, counting from the 3′-end of the sense strand;
the internal positions exclude a cleavage site region of the antisense strand, optionally wherein the internal positions include all positions except positions 12-14, counting from the 5′-end of the antisense strand; and/or
the internal positions include all positions except positions 11-13 on the sense strand, counting from the 3′-end, and positions 12-14 on the antisense strand, counting from the 5′-end;
(ii) the one or more lipophilic moieties are conjugated to one or more of the internal positions selected from the group consisting of positions 4-8 and 13-18 on the sense strand, and positions 6-10 and 15-18 on the antisense strand, counting from the 5′end of each strand, optionally wherein the one or more lipophilic moieties are conjugated to one or more of the internal positions selected from the group consisting of positions 5, 6, 7, 15, and 17 on the sense strand, and positions 15 and 17 on the antisense strand, counting from the 5′-end of each strand; and/or
(iii) the internal positions in the double stranded region exclude a cleavage site region of the sense strand.
6 . The dsRNA agent claim 4 , wherein the sense strand is 21 nucleotides in length, the antisense strand is 23 nucleotides in length, and the lipophilic moiety is conjugated to position 21, position 20, position 15, position 1, position 7, position 6, or position 2 of the sense strand or position 16 of the antisense strand, optionally wherein:
the lipophilic moiety is conjugated to position 21, position 20, position 15, position 1, or position 7 of the sense strand;
the lipophilic moiety is conjugated to position 21, position 20, or position 15 of the sense strand;
the lipophilic moiety is conjugated to position 20 or position 15 of the sense strand; or
the lipophilic moiety is conjugated to position 16 of the antisense strand.
7 . The dsRNA agent of claim 4 , wherein the lipophilic moiety is an aliphatic, alicyclic, or polyalicyclic compound, optionally wherein:
the lipophilic moiety is selected from the group consisting of lipid, cholesterol, retinoic acid, cholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, 1,3-bis-O (hexadecyl) glycerol, geranyloxyhexyanol, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl) lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine; and/or
the lipophilic moiety contains a saturated or unsaturated C4-C30 hydrocarbon chain, and an optional functional group selected from the group consisting of hydroxyl, amine, carboxylic acid, sulfonate, phosphate, thiol, azide, and alkyne, optionally wherein the lipophilic moiety contains a saturated or unsaturated C6-C18 hydrocarbon chain or a saturated or unsaturated C16 hydrocarbon chain, optionally wherein the C16 hydrocarbon chain is conjugated to position 6, counting from the 5′-end of the strand.
8 . The dsRNA agent of claim 4 , wherein:
the lipophilic moiety is conjugated via a carrier that replaces one or more nucleotide(s) in the internal position(s) or the double stranded region, optionally wherein the carrier is a cyclic group selected from the group consisting of pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3] dioxolanyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuranyl, and decalinyl; or is an acyclic moiety based on a serinol backbone or a diethanolamine backbone;
the lipophilic moiety is conjugated to the double-stranded iRNA agent via a linker containing an ether, thioether, urea, carbonate, amine, amide, maleimide-thioether, disulfide, phosphodiester, sulfonamide linkage, a product of a click reaction, or carbamate;
the lipophilic moiety is conjugated to a nucleobase, sugar moiety, or internucleosidic linkage;
the lipophilic moiety is conjugated via a bio-clevable linker selected from the group consisting of DNA, RNA, disulfide, amide, funtionalized monosaccharides or oligosaccharides of galactosamine, glucosamine, glucose, galactose, mannose, and combinations thereof;
the 3′ end of the sense strand is protected via an end cap which is a cyclic group having an amine, said cyclic group being selected from the group consisting of pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3] dioxolanyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuranyl, and decalinyl; and/or
the dsRNA agent further comprises a targeting ligand that targets a neuronal cell, optionally wherein the targeting ligand is a GalNAc conjugate.
9 . The dsRNA agent of claim 1 , wherein the hydrophobicity of the double-stranded RNA agent, measured by the unbound fraction in a plasma protein binding assay of the double-stranded RNA agent, exceeds 0.2, optionally wherein the plasma protein binding assay is an electrophoretic mobility shift assay using human serum albumin protein.
10 . The dsRNA agent of claim 1 , wherein the dsRNA agent comprises at least one modified nucleotide, optionally wherein:
(i) no more than five of the sense strand nucleotides and no more than five of the nucleotides of the antisense strand are unmodified nucleotides, or all of the nucleotides of the sense strand and all of the nucleotides of the antisense strand are modified nucleotides; and/or
(ii) at least one of the modified nucleotides is selected from the group a deoxy-nucleotide, a 3′-terminal deoxy-thymine (dT) nucleotide, a 2′-O-methyl modified nucleotide, a 2′-fluoro modified nucleotide, a 2′-deoxy-modified nucleotide, a locked nucleotide, an unlocked nucleotide, a conformationally restricted nucleotide, a constrained ethyl nucleotide, an abasic nucleotide, a 2′-amino-modified nucleotide, a 2′-O-allyl-modified nucleotide, 2′-C-alkyl-modified nucleotide, 2′-hydroxly-modified nucleotide, a 2′-methoxyethyl modified nucleotide, a 2′-O-alkyl-modified nucleotide, a morpholino nucleotide, a phosphoramidate, a non-natural base comprising nucleotide, a tetrahydropyran modified nucleotide, a 1,5-anhydrohexitol modified nucleotide, a cyclohexenyl modified nucleotide, a nucleotide comprising a 5′-phosphorothioate group, a nucleotide comprising a 5′-methylphosphonate group, a nucleotide comprising a 5′ phosphate or 5′ phosphate mimic, a nucleotide comprising vinyl phosphonate, a nucleotide comprising adenosine-glycol nucleic acid (GNA), a nucleotide comprising thymidine-glycol nucleic acid (GNA) S-Isomer, a nucleotide comprising 2-hydroxymethyl-tetrahydrofurane-5-phosphate, a nucleotide comprising 2′-deoxythymidine-3′phosphate, a nucleotide comprising 2′-deoxyguanosine-3′-phosphate, and a terminal nucleotide linked to a cholesteryl derivative and a dodecanoic acid bisdecylamide group; and combinations thereof, optionally wherein:
(a) the modified nucleotide is selected from the group consisting of a 2′-deoxy-2′-fluoro modified nucleotide, a 2′-deoxy-modified nucleotide, 3′-terminal deoxy-thymine nucleotides (dT), a locked nucleotide, an abasic nucleotide, a 2′-amino-modified nucleotide, a 2′-alkyl-modified nucleotide, a morpholino nucleotide, a phosphoramidate, and a non-natural base comprising nucleotide;
(b) the modified nucleotide comprises a short sequence of 3′-terminal deoxy-thymine nucleotides (dT); and/or
(c) the modifications on the nucleotides are 2′-O-methyl, GNA and 2′fluoro modifications.
11 . The dsRNA agent of claim 1 , further comprising at least one phosphorothioate internucleotide linkage, optionally wherein the dsRNA agent comprises 6-8 phosphorothioate internucleotide linkages.
12 . The dsRNA agent of claim 1 , wherein each strand is no more than 30 nucleotides in length, optionally wherein:
at least one strand comprises a 3′ overhang of at least 1 nucleotide;
at least one strand comprises a 3′ overhang of at least 2 nucleotides;
the double stranded region is 15-30, 17-23, 17-25, 23-27, 19-21, or 21-23 nucleotide pairs in length; and/or
each strand has 19-30, 19-23, or 21-23 nucleotides.
13 . The dsRNA agent of claim 1 , further comprising:
(i) a terminal, chiral modification occurring at the first internucleotide linkage at the 3′ end of the antisense strand, having the linkage phosphorus atom in Sp configuration,
a terminal, chiral modification occurring at the first internucleotide linkage at the 5′ end of the antisense strand, having the linkage phosphorus atom in Rp configuration, and
a terminal, chiral modification occurring at the first internucleotide linkage at the 5′ end of the sense strand, having the linkage phosphorus atom in either Rp configuration or Sp configuration;
(ii) a terminal, chiral modification occurring at the first and second internucleotide linkages at the 3′ end of the antisense strand, having the linkage phosphorus atom in Sp configuration,
a terminal, chiral modification occurring at the first internucleotide linkage at the 5′ end of the antisense strand, having the linkage phosphorus atom in Rp configuration, and
a terminal, chiral modification occurring at the first internucleotide linkage at the 5′ end of the sense strand, having the linkage phosphorus atom in either Rp or Sp configuration;
(iii) a terminal, chiral modification occurring at the first, second and third internucleotide linkages at the 3′ end of the antisense strand, having the linkage phosphorus atom in Sp configuration,
a terminal, chiral modification occurring at the first internucleotide linkage at the 5′ end of the antisense strand, having the linkage phosphorus atom in Rp configuration, and
a terminal, chiral modification occurring at the first internucleotide linkage at the 5′ end of the sense strand, having the linkage phosphorus atom in either Rp or Sp configuration;
(iv) a terminal, chiral modification occurring at the first, and second internucleotide linkages at the 3′ end of the antisense strand, having the linkage phosphorus atom in Sp configuration,
a terminal, chiral modification occurring at the third internucleotide linkages at the 3′ end of the antisense strand, having the linkage phosphorus atom in Rp configuration,
a terminal, chiral modification occurring at the first internucleotide linkage at the 5′ end of the antisense strand, having the linkage phosphorus atom in Rp configuration, and
a terminal, chiral modification occurring at the first internucleotide linkage at the 5′ end of the sense strand, having the linkage phosphorus atom in either Rp or Sp configuration; or
(v) a terminal, chiral modification occurring at the first, and second internucleotide linkages at the 3′ end of the antisense strand, having the linkage phosphorus atom in Sp configuration,
a terminal, chiral modification occurring at the first, and second internucleotide linkages at the 5′ end of the antisense strand, having the linkage phosphorus atom in Rp configuration, and
a terminal, chiral modification occurring at the first internucleotide linkage at the 5′ end of the sense strand, having the linkage phosphorus atom in either Rp or Sp configuration.
14 . The dsRNA agent of claim 1 , wherein:
the dsRNA further comprises a phosphate or phosphate mimic at the 5′-end of the antisense strand, optionally wherein the phosphate mimic is a 5′-vinyl phosphonate (VP);
the base pair at the 1 position of the 5′-end of the antisense strand of the duplex is an AU base pair; and/or
the sense strand has a total of 21 nucleotides and the antisense strand has a total of 23 nucleotides.
15 . A pharmaceutical composition for inhibiting expression of a gene encoding ADRA2A, comprising the dsRNA agent of claim 1 , optionally wherein:
the pharmaceutical composition further comprises a lipid formulation;
the dsRNA agent is in an unbuffered solution, optionally wherein the unbuffered solution is saline or water; and/or
the dsRNA agent is in a buffer solution, optionally wherein the buffer solution comprises acetate, citrate, prolamine, carbonate, or phosphate or any combination thereof, or the buffer solution is phosphate buffered saline (PBS).
16 . An isolated cell containing the dsRNA agent of claim 1 .
17 . A kit or a container comprising the dsRNA agent of claim 1 or a pharmaceutical composition comprising the dsRNA agent of claim 1 , optionally wherein the container is a vial, a syringe, or an intrathecal pump.
18 . A method of inhibiting expression of an ADRA2A gene in a cell, the method comprising contacting the cell with the dsRNA agent of claim 1 , or a pharmaceutical composition comprising the dsRNA agent of claim 1 , thereby inhibiting expression of the ADRA2A gene in the cell.
19 . The method of claim 18 , wherein:
(i) the cell is within a subject;
optionally wherein the subject is human;
optionally wherein the subject has an ADRA2A-associated disorder;
optionally wherein the ADRA2A-associated disorder is a neurodegenerative disorder,
optionally wherein the neurodegenerative disorder is a tauopathy, Alzheimer's disease, or a primary tauopathy; and/or
optionally wherein the primary tauopathy is selected from the group of disorders consisting of: frontotemporal dementia (FTD), frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17), frontotemporal lobar degeneration (FTLD), progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), chronic traumatic encephalopathy (CTE), Pick's disease (PiD), globular glial tauopathies (GGTs), argyrophilic grain disease (AGD), and primary age-related tauopathy (PART);
(ii) contacting the cell with the dsRNA agent inhibits the expression of ADRA2A by at least 30%; and/or
(iii) inhibiting expression of ADRA2A decreases ADRA2A protein level in serum of the subject by at least 30%.
20 . A method of treating a subject having a disorder that would benefit from reduction in ADRA2A expression, or preventing at least one symptom in the subject, the method comprising administering to the subject a therapeutically effective amount of the dsRNA agent of claim 1 , or a pharmaceutical composition comprising the dsRNA agent of claim 1 , thereby treating the subject having the disorder that would benefit from reduction in ADRA2A expression, optionally wherein:
the disorder is an ADRA2A-associated disorder, optionally wherein the ADRA2A-associated disorder is selected from the group consisting of Alzheimer's disease, a primary tauopathy, frontotemporal dementia (FTD), frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17), frontotemporal lobar degeneration (FTLD), progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), chronic traumatic encephalopathy (CTE), Pick's disease (PiD), globular glial tauopathies (GGTs), argyrophilic grain disease (AGD), and primary age-related tauopathy (PART);
the subject is human;
the administration of the agent to the subject causes a decrease in ADRA2A protein accumulation;
the dsRNA agent is administered to the subject at a dose of about 0.01 mg/kg to about 50 mg/kg;
the dsRNA agent is administered to the subject intrathecally;
the method further comprises determining the level of ADRA2A in a sample(s) from the subject, optionally wherein the level of ADRA2A in the subject sample(s) is an ADRA2A protein level in a blood, serum, or cerebrospinal fluid sample(s); and/or
the method further comprises administering to the subject an additional therapeutic agent.